The micro titer plate reader has been a workhorse for bioanalytical testing for decades. It enables the facile and rapid interrogation of an array of chemical reactions. Typically 8 by 12 well formats of 96 wells are filled with reagents and or products of a calorimetric or fluorescent reaction. Higher order formats include multiples of 96 wells. Such micro-titer plates are exposed to light of a desired wavelength and the interaction of the reacting species with the light is recorded.
Reactions may be immunochemical, enzyme based, polymerizations, intercollations or any of the varied molecular biological and biochemical systems investigated in the biochemists' laboratory. The interactions may include and are not limited to absorbance, transmittance, scatter and fluorescence. Micro titer plate readers are designed to generate one or more wavelengths of interest. Typically the light is generated using a wide spectrum source, arc lamps and halogen bulbs with numerous filters or gratings are common design components.
Biochemical reactions formatted in either homogeneous or heterogeneous based detection platforms are also performed in miniaturized systems, e.g. on micro fluidic chips and micro arrays. In miniaturized systems, reaction volumes are contained within channels and/or with carefully modified surface chemistry to allow for a plethora of chemical and biological analysis in small reaction volumes. The chemistry is typically interrogated using confocal microscopy or imaging to assess the extent of higher density features. Tens to hundreds of thousands of enzyme, immunochemical, nucleic acid and protein reactions can be followed simultaneously. Lamp and lasers power the various detection systems designed to report on the extent of reaction.
One now commonplace procedure performed in the bioanalytical laboratory is the polymerase chain reaction (PCR). The technique has become fundamental to molecular biology. It is one of a family of methods (i.e. reverse transcriptase PCR) for synthesizing a given quantity of pre-selected biopolymer. PCR functions on DNA. In a typical experiment, the DNA of interest is separated into two complementary strands. The ends of each strand bind to a primer at the end where the synthesis begins. The addition of the DNA polymerase initiates the synthesis of a complementary strand on each single strand creating a doubling of the amount of DNA. The process is repeated until a sufficient number of DNA segments have been synthesized. A unique temperature profile is used to advance the reaction through the phases of separation (melting), primer binding (annealing), and replication (extension). While the PCR technique has become a workhorse of the biotechnologist due to the enhanced sensitivity it offers over blotting techniques, PCR is not ideally suited to quantitation. Small differences between sample sizes can become huge differences in final amplified material after multiple doublings.
A typical PCR reaction can be seen in three phases: an early lag phase, an exponential growth phase and a plateau region. The lag phase is a consequence of the sensitivity of the instrument and the background signal of the probe system used to detect the PCR product. The exponential growth phase commences when sufficient product has accumulated to be detected by the specific instrument. During this exponential growth the amplification is described by Tn=Ti(E)n, where Tn is the amount of target sequence at cycle n, Ti is the initial amount of target material, and E is the efficiency of amplification. In the final plateau phase the amplification efficiency drops as product competes more effectively with primers for annealing and the amount of enzyme becomes limiting. The exponential equation no longer holds in the plateau phase. Most of the quantitative information is found in the exponential cycles but the exponential cycles typically comprise only 4 or 5 cycles out of 40. For traditional PCR methods, identifying the exponential cycles requires the reaction be split into multiple reaction tubes that are assayed for PCR product after varying numbers of cycles. This requires either assaying many tubes or having a fairly good idea of the answer before the experiment is begun. Once the position of this phase is determined the experimental phase can be compared to known standards and the copy number can be calculated.
Instrumentation advancements have made real-time monitoring of PCR reactions possible. Thermocycling is carried out using standard techniques known to those skilled in the art, including rapid cycling PCR. Fluorescence monitoring at each cycle for quantitative PCR was developed by Higuchi et al., “Simultaneous Amplification and Detection of Specific DNA Sequences,” Bio. Technology, 10:413-417, 1992, which is herein expressly incorporated by reference in its entirety. Ethidium bromide was used as the fluorescent entity. Fluorescence was acquired once per cycle for a relative measure of product concentration. The cycle where observable fluorescence first appeared above the background fluorescence correlated with the starting copy number, allowing the construction of a standard curve. Alternatively PCR amplification may be conducted with fluorescently labeled hybridization probes. The hybridization probe system comprises two oligonucleotide probes that hybridize to adjacent regions of a DNA sequence wherein each oligonucleotide probe is labeled with a respective member of a fluorescent energy transfer pair. In this embodiment, the presence of the target nucleic acid sequence in a biological sample is detected by measuring fluorescent energy transfer between the two-labeled oligonucleotides. A number of strategies now exist using molecular beacons or intercollating dyes all of which are strategies to increase signal as a function of increasing DNA concentration as the synthesis cycles increase.
Such instrumentation and fluorescent monitoring techniques have made kinetic PCR significantly easier than traditional competitive PCR. The ease, accuracy and precision of quantitative PCR have all improved by allowing observation of the PCR product concentration at every cycle. In the Roche® Diagnostics embodiment of the kinetic PCR instrument, PCR reactions are conducted using the Light Cycler®, a real-time PCR instrument that combines a rapid thermal cycler with a fluorimeter. Through the use of such a device, PCR product is detected with fluorescence and no additional sample processing, membrane arrays, gels, capillaries, or other analytical tools are necessary. Other PCR instrumentation as known in the art may be used in the practice of the present invention.
Separation by electrophoresis is based on differences in solute velocity in an electric field. The velocity of a charged analyte is a function of its electrophoretic mobility and the applied voltage. The method of electrophoresis is used in a number of different techniques including capillary gel electrophoresis, capillary zone electrophoresis, micellar electrokinetic chromatography, capillary electro chromatography, isotachophoresis and isoelectric focusing.
In general, the mobility of an analyte in a particular medium is constant and characteristic of that analyte. The analytes mobility is a result of two factors. The analyte is attracted to the electrode of opposite charge, pulling it through the medium. At the same time, however, frictional forces try to prevent the analyte moving toward the charge. The balance of these forces determines the actual overall mobility of the analyte. An analytes size, polarity and number of electric charge(s), relative hydrophobicity and ionic strength determine how rapidly an electric field can move the analyte through a medium. A buffer is used to assist the flow of the analyte relative to the field. The buffer's chemical composition, pH, temperature and concentration alter the mobility of the analyte. Many important biological molecules such as amino acids, peptides, proteins, nucleotides, and nucleic acids, posses ionizable groups and, therefore, at any given pH, exist in solution as electrically charged species either as cations containing a positive (+) charge or as anions containing a negative (−) charge. Depending on the nature of the net charge, the charged particles will migrate either to the cathode or to the anode. A small analyte will have less frictional drag than a large analyte and hence move through the medium faster than a large analyte. Similarly, a multiply charged analyte will experience more attraction to the electrode and also move through the medium faster than a singly charged analyte. It is this difference in solute velocities that is responsible for the separating effect in electrophoresis that results in resolution of the species detected.
Gel electrophoresis is a method that separates molecules such as DNA or proteins on the basis of their physical properties. A gel is a solid colloid. Thus, gel electrophoresis refers to the technique in which molecules are forced to cross a span of gel, motivated by an electrical current. Activated electrodes at either end of the gel provide the electric field and thus the driving force for the migration of the analyte. During electrophoresis, molecules are forced to move through the pores in the gel when the electrical current is applied. Their rate of migration, through the induced electric field, depends on the strength of the field, their charge, their size and the shape of the molecules, the relative hydrophobicity of the molecules, and on the ionic strength and temperature of the buffer in which the molecules are moving.
One use of gel electrophoresis is the identification of particular DNA molecules by the band patterns they yield in gel electrophoresis, after being cut with various restriction enzymes. Viral DNA, plasmid DNA, and particular segments of chromosomal DNA can all be identified in this way. Another use is the isolation and purification of individual DNA fragments containing interesting genes, which can be recovered from the gel with full biological activity.
Capillary Zone Electrophoresis (CZE) replaces the gel in gel electrophoresis with the combination of a buffer and a solid support contained within the capillary. In CZE, the analyte must move through the solid support contained within the capillary under the action of the buffer, which is charged by the applied electric field. The buffer's chemical nature, pH, temperature, concentration and the presence of surfactant additives can be selected to assist in fully resolving (i.e., spatially separating different analytes in the capillary with respect to the time from introduction of the sample) different analytes in space (position in the capillary) with respect to time. Analytes separated by CZE can be detected based on absorption or fluorescence. Detection can be carried out using on-column or fiber optic Z-cells.
In addition to electrophoretic techniques, separation of molecules can be carried out in the absence of an applied field using chromatographic techniques. In liquid chromatography, the molecule dissolved in a buffer can still be charged, but rather than an electric field creating the driving force, molecule migration is dependent on the flow of the buffer. Frictional forces due to the interaction of the molecule with a solid support present in a column, act to prevent the molecule from moving with the buffer. The molecule's size, hydrophobicity, and ionic strength determine how rapidly the buffer can move the molecule through a medium. The buffer's chemical composition, pH, temperature and concentration together with the nature of the solid support dispersed in the column alter the mobility of the molecule. High performance liquid chromatography (HPLC) utilizes pumps to increase the flow of buffer through the columns resulting in high column backpressure, improved resolution, increased flow rates and reduced analysis times. By reducing the diameter of the column and/or increasing the length of the column the resolution can be improved. However, a problem with narrower columns (milli bore or micro bore) involves detection of the eluted species. As the diameter of the capillary in the narrow bore HPLC systems is further reduced, only a small number of molecules are available for detection in a small-defined area.
Microfluidic systems comprised of microfluidic chips, automated reagent delivery apparatus and detection instrumentation are designed to minimize the users' effort in reagent delivery, reagent dilution and/or mixing, initiating chemical reactions and detecting those chemical reactions in small volumes within highly automated environments. Among the numerous applications that exist, fluorescence is a commonly used detection format. It is a sensitive and robust method for detecting enzyme assays, immunoassays, polymerase chain reaction (PCR), quantitative PCR, genomic sequencing among many other important chemical reactions. Both homogeneous and heterogeneous reactions are suited to such devices and analysis is not limited by whether the reaction takes place in free solution or on a solid support or within a narrow pore. Often microfluidic devices are produced by etching, molding or embossing channels and wells into solid substrates (glass, silicon, plastic, etc.). Numerous layers of the device can be fabricated and then the layers assembled to form the final analysis tool. Channels can be etched in single or multiple dimensions enabling more complicated chemical separation and detection. Such devices can be used to introduce reagents directly onto the chip or interfaced with automation equipment for such purposes. Like all fluorogenic detection, these systems require an excitation source.